Project Summary We wish to understand two important processes that sustain human vision; the visual cycle is responsible for regeneration of photopigment bleached by absorption of visible light and cellular metabolism is required in every living cell to provide energy. To study these processes, we need a method to visualize individual cells and measure molecular dynamics in the living eye. Some of the molecules involved are intrinsically fluorescent, but are inaccessible in the living eye with single- photon fluorescence imaging because the excitation falls outside the range of radiation that can penetrate the optics of the eye. By using considerably longer excitation wavelengths, two-photon excited fluorescence imaging has the potential to excite these otherwise inaccessible fluorophores and provide intrinsic contrast for imaging a number of retinal structures. In our initial experiments, we used an adaptive optics scanning light ophthalmoscope (AOSLO) to image two-photon fluorescence from cone inner segments in the living macaque eye (Hunter et al, 2011). By correcting the longitudinal chromatic aberration and material dispersion of the eye, we plan to improve the efficiency of our imaging systems and develop methodology for reliably imaging both structure and function of multiple retinal layers in the eye. Not only will this capability provide insight into normal cell mosaics and their biochemical processes, it will also improve our understanding of many diseases that impact these retinal biochemical cascades such as Stargardt's disease, macular degeneration and Leber's hereditary optic neuropathy.